Enzymatic polymerization 446 Subject

Poly(3-hydroxya]kanoate)s (PHAs) are natural polyesters, which many organisms in the environment accumulate in the form of intracellular granules to store carbon and energy when they are subjected to stress conditions [1-3]. PHAs are produced by a fermentation process in the bacteria by means of enzymatic polymerization (PHA synthase). The type of biosynthesized polymers is determined by the substrate specificities of the PHAs synthases and depends on the carbon source. PHAs are semi-crystalline, isotactic (only the enantiomer of absolute configuration R is present in these polymers) with a hydrophobic character. Although the most well-studied PHA is poly(3-hydroxybutyrate) (PHB), over 140 constitutive monomer units [4] have been investigated. 

What reasons are there for mixing polymerizable lipids with natural ones Polymerized membrane systems, especially those based on diacetylenic lipids, have proven to be excessively rigid and to show no phase transition. Addition of natural lipids could help to retain a certain membrane mobility even in the polymerized state, with almost unaffected stability. Furthermore, natural lipids can provide a suitable environment for the incorporation of membrane proteins into polymerizable membranes (see 4.2.3). Besides this, enzymatic hydrolysis of the natural membrane component can be used for selectively opening up a vesicle in order to release entrapped substances in a defined manner (see 4.2.2). Therefore, it is interesting to learn about the miscibility of polymerizable and natural lipids and also about the polymerization behavior of these mixtures. Investigations on this subject have thus far focused on mixtures of natural lipids with polymerizable lipids carrying diacetylene moieties. 

Peroxidase-catalyzed polymerization of various flavonoids was investigated in an equivolume mixture of 1,4-dioxane and pH 8 buffer [110]. Flavonols (quercetin) as well as isoflavones (diadzein and 5,6,4 -trihydroxyisoflavone) were subjected to the enzymatic oxidative polymerization to produce the flavonoid polymers. From diadzein, the polymer soluble in DMF and DMSO was formed in a high yield. 

An entirely different approach to sugar-based polymers involves the use of selective enzymatic catalysts to prepare vinyl sugar monomers that are subsequently polymerized via chemical catalysts. Tokiwa and Kitagawa (25) published extensively on this subject, and their contribution within this book describes a wide range of sugar monomer structures. 

Monomer 7 synthesized via conventional organic technique, was subjected to the Bacillus sp. derived chitinase-catalyzed polymerization. Without enzyme, the monomer was gradually decomposed by non-enzymatic hydrolysis from aqueous media. In contrast, the ring opening reaction of oxazoline monomer was drastically activated by chitinase, giving rise to polymer 8 within 0.7 h. During the polymerization, the reaction mixture was kept homogeneous. 

Complex mixtures are produced by non-enzymatic browning reactions between thermally oxidized lipids and amines, amino acids and proteins (see Chapter 11.B.4). Interactions between aldehydes, epoxides, hydroxy ketones, and dicarbonyls with proteins cause browning that has been related with losses of lysine, histidine, and methionine. Schiff base formation results in polymerization to form brown macromolecules. Interactions between epoxyalkenals formed at elevated temperatures and reactive groups of proteins produce protein pyrroles polymers and volatile heterocyclic compounds. Much of the published research in this complex chemical area was based on model systems. More stmctural information is needed however with real foods subjected to frying conditions. 

Medium size lactones, 3-valerolactone (5-VL, six-membered) and e-caprolactone (e-CL, seven-membered), were subjected to lipase-catalyzed polymerizations. Lipases CC, PF, and PPL showed high catalytic activity for the polymerization of S-VL (149,150). The molecular weight of enzymatically obtained... 

The potentiostatic multi-pulse potentiometry described here allows the dynamic measurement of potentials. The advantages of this method are the short time required for the analysis and the low noise of the signal. The "ancestor" of this technique, enzyme chronopotentiometry [7 ], posed problems of reproducibility when it was applied to the immobilized redox polymer. The excellent reproducibility of our method is clearly shown in fig. 3b. These techniques were the fundamental developments to conceive redox-FETs for the first time. After immobilization of NAD -dependent dehydrogenases covalently on the surface of the transducer the enzymatically produced NADH would be catalytically oxidized in situ by the polymeric mediator. To this very compact combination the substrate and NAD+ as cosubstrate have to be applied externally. The coimmobilization of the coenzyme NAD+ would lead to reagentless sensors. This is a subject of forthcoming investigations... 

All the polymeric NAD(P)+-derivatives have been checked for their cofactor activity and compatibility with enzymatic biocatalytic processes. The polymer-linked NAD(P)" -derivatives were associated with NAD(P) -dependent enzymes such as AlDH [281, 282, 291, 292], lactate dehydrogenase [286, 292], malate dehydrogenase [288, 292] and aldehyde dehydrogenase [287]. It was found that different NAD(P)+-polymers are active as cofactors towards different enzymes. For example, polyethyleneimine and polylysine bound NAD+-derivative revealed 60% and 25% activity, respectively, as compared with the native NAD+ in the presence of rabbit muscle lactate dehydrogenase, but only minute activity (ca. 2-7%) in the presence of alanine dehydrogenase from Bacillus suhtilis [281]. A comparative study of the cofactor activity with different enzymes is a subject of great interest. Even though several studies [279] attempt to predict the structural/functional relationship for the polymer-bound NAD(P)+-derivatives,... 

The concept of TSAS was further proven to be vahd by the following experiments. Chi-oxa monomer was subjected to polymerization by enzymatic catalysis. The hydrolysis enzymes used were chitinase (family 18) involving an oxazohnium intermediate and lysozyme involving an oxocarbenium ion intermediate (Fig. 13). With the former enzyme, synthetic chitin was quantitatively obtained after 50 h at pH 10.6, whereas with the latter no chitin was produced after 165 h of reaction [69]. This imphes that the oxazoline monomer could not be a substrate for the lysozyme enzyme. 

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